ABSTRACT: Current antiretroviral therapy (ART) does not eradicate HIV as shown by the rapid rebound of viremia upon treatment interruption. Rescuing viral gene expression in latently infected cells by compounds termed Latency Reversing Agents (LRA) is a strategy to reduce the virus reservoir in ART-treated HIV-infected individuals (i.e., shock & kill). HIV latency is triggered by mechanisms that lead to the silencing of virus expression including epigenetic DNA modification through histone methylation and deacetylation, limited availability of critical transcription factors and inefficient elongation of the nascent viral transcripts. Recent studies in ART-treated SIV- infected macaques demonstrated that depletion of CD8+ lymphocytes is followed by reactivation of virus production, and increased susceptibility to the LRA effect of the IL-15 superagonist ALT803. These results strongly suggest that CD8+ lymphocyte play an important yet understudied role in silencing HIV expression. In this proposal, we hypothesize that CD8+ T cells suppress HIV gene expression by promoting the establishment and maintenance of HIV latency, and that reversal of this effect may result in a significant amplification of the LRA effect of various compounds, thus impacting the reservoir size and stability in vivo. We will use innovative approaches to identify the CD8+ T cell mediated mechanisms of HIV silencing. In Aim 1, we will employ a novel model of HIV latency to characterize the role of CD8+ T cells in the establishment of HIV latency. We will quantify HIV gene expression in CD4+ T cell subsets and characterize the impact of CD8+ T cells on HIV latency establishment. In Aim 2, we will characterize the role of CD8+ T cells in the maintenance of HIV latency. Our in vitro model will allow us to define how CD8+ T cells maintain HIV latency during cytokine driven activation, proliferation or differentiation. In Aim 3, we will design a combinatorial anti-latency strategy that will eliminate most if not all latently infected cells. We will employ high dimensional flow cytometry to define alterations in phenotype associated with suppression and activation of latency reversal. Ultimately, we will characterize the role of CD8+ lymphocyte in counter-acting the latency reversal by specific LRAs (and LRA combinations) as a key step to develop effective interventions in which viral transcription in latently infected cells is rescued to reduce the reservoir size. The experiments described in this proposal will identify novel mechanisms of HIV silencing that can lead to the reactivation and subsequent elimination of most (if not all) latently infected cells. We propose that this overarching goal can be achieved through a step-wise scientific approach that includes in vitro and ex vivo studies that will address a newly-defined mechanism of HIV persistence. We believe that the work proposed in this application will allow us to understand the mechanisms by which CD8+ lymphocytes suppress virus transcription and ultimately promote HIV latency and persistence in ART-treated HIV-infected individuals, thus helping designing new approaches for HIV eradication.